Nucleic acids, as diagnostic targets, are of great importance in clinical settings. For many years, DNA has been successfully used as a molecular target for the diagnosis of many diseases, including prenatal conditions (Lun et al., Proc. Natl. Acad. Sci. USA 105(50): 19920-5, 2008), cancer (Gormally et al., Mutat. Res. 635(2-3): 105-17, 2007) and infectious diseases (Hawkes and Kain, Expert Rev. Anti Infect. Ther. 5(3): 485-95, 2007). DNA-based diagnostics include polymerase chain reaction (PCR) (Rougemont et al., J. Clin. Microbiol. 42(12): 5636-43, 2004), loop-mediated isothermal amplification (LAMP) (Hopkins et al., J. Infect. Dis. 208(4); 645-652, 2013), etc., which all offer sensitive detection of target DNA. However, they largely rely on extraction of DNA before detection, and the extraction process can be laborious and prone to contamination. This is particularly problematic in settings where large numbers of samples are being processed.
RNA is also a good target for diagnostics (Miura et al., Clin. Med. Oncol. 2: 511-27, 2008; Ng et al., Clin. Chem. 48(8): 1212-7, 2002; Skog et al., Nat. Cell Biol. 10(12): 1470-U209, 2008; Murphy et al., Amer. J. Trop. Med. Hyg. 86(3): 383-94, 2012; Mens et al., Malar. J. 5: 80, 2006; Mitra et al., Front. Genet. 3: 17, 2012). Current RNA detection methods primarily include microarray hybridization, reverse transcription PCR, nucleic acid sequence-based amplification (NASBA) and RNA hybridization assays. Microarray assays are capable of measuring the expression levels of large numbers of genes simultaneously or genotyping multiple regions of a genome in a single RNA sample. Yet when only a small number of genes are required to be tested in a large number of samples, as in clinical diagnostic settings, the use of microarrays becomes impractical, as the cost-effectiveness is low and labor demand is high. Reverse transcription PCR is currently the most widely used technique for RNA quantification, but as it depends on purification and reverse transcription of RNA, the accuracy and reproducibility of quantification can be reduced by varied efficiencies of extraction and reverse transcription processes (Bustin and Nolan, J. Biomol. Tech. 15(3): 155-66, 2004).
NASBA (Schneider et al., J. Clin. Microbiol. 43(1): 402-5, 2005) and size-coded ligation-mediated polymerase chain reaction (SL-PCR) (Arefian et al., Nucleic Acids Res. 39(12), 2011) detect RNA without prior reverse transcription, enabling specific, sensitive, quantitative detection of RNA. However, they both still rely on extraction of RNA and decontamination of DNA, which are expertise-demanding and error-prone processes (Peirson and Butler, Methods Mol. Biol. 362: 315-27, 2007).
A hybridization-based RNA detection technique, previously developed by the inventor, avoided RNA purification and reverse transcription, measuring RNA levels sensitively and specifically in whole blood with high throughput (Zheng et al., Clin. Chem. 52(7): 1294-302, 2006). Although capable of multiplex detection, this method requires specially-made, branched DNA multimers as signal amplifiers, which hinders its application in ordinary laboratory settings.
Ligation-dependent PCR assay (Hsuih et al., J. Clin. Microbiol. 34(3): 501-7, 1996) also detects RNA without the need for RNA extraction or reverse transcription. The assay uses two DNA capture probes for RNA isolation and two DNA hemiprobes for subsequent PCR. The DNA capture probes have a target-complementary sequence as well as a biotin moiety, which can bind to a surface with streptavidin. The two DNA hemiprobes are designed to bind to target RNA in juxtaposition to one another. Target RNA is directly purified from sample lysate by capture probes anchored to a solid surface through the interaction between biotin and streptavidin. The hemiprobes can then be linked to each other by incubation with a ligase (see EP1311703) to form a full probe that serves as a template for PCR.
All references discussed herein are incorporated by reference in their entirety.